Pcr8 gw topo ta cloning kit

Inserts for cloning were produced by PCR on cDNA from urediospore RNA using nested PCR. First-round PCR was in 10-µl reactions with 1 µl 10× buffer, 1 µl 10 mM dNTP mix, 0.5 µl 0.1 µM f1 primers each (see Table S1), 1-µl cDNA, and 0.1 µl Long PCR Enzyme Mix (Thermo Fischer Scientific). Second round PCR was in 50-µl reactions with 5 µl 10× buffer, 5 µl 10 mM dNTP mix, 2.5 µl f2 primers each, 2 µl PCR product from the first round reaction, and 0.5 µl Long PCR Enzyme Mix. Cycling conditions were the same for both PCRs: initial denaturation 3 min 94°C, then 35 cycles with 20 s 95°C, 30 s 55°C, and 60 s 68°C; final elongation 7 min 68°C. Second round PCR products were purified using the peqGOLD Cycle-Pure Kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) and inserted into the pCR^™8/GW/TOPO^® plasmid using the pCR^™8/GW/TOPO TA Cloning^® Kit (Invitrogen, Life Sciences, Carlsbad, CA, USA), following the instructions of the manufacturer and using 4 µl of the purified PCR product. All constructs were verified by sequencing. For production of constructs without the signal peptide, the inserts were PCR amplified again using a third set of primers and again inserted into the pCR^™8/GW/TOPO^® plasmid. All three sets of primers are supplied in Table S1.

The RNA interference (RNAi) vector was constructed by PCR amplification of a 291 bp fragment from OsISA1 and a 206 bp fragment from OsBEIIa cloned from Hwacheong cDNA. These fragments were subcloned into pDONR201 (Invitrogen, USA), and then transferred into the RNAi vector pH7GWIWG (II) using Gateway BP and LR clonase enzyme mixes (Invitrogen, USA). The full-length OsBEIIa cDNA was amplified from Hwacheong cDNA and used for constructing the overexpression vector. The amplified fragment was transferred into pMDC32 via pCR™ 8/GW/TOPO® TA Cloning Kit (Invitrogen, USA). The RNAi constructs were transformed into wild-type Dongjin (a japonica cultivar), and the overexpression construct was transformed into callus of the sug-h mutant. Transformation was performed using a modification of the previously published Agrobacterium-mediated transformation method (Nishimura et al. 2006 (link)).

The CYP71AZ1 open reading frame was PCR-amplified using a high-fidelity DNA polymerase (PrimeSTAR Max; Clontech) and primers encoding an additional 6xHis tag at the 3′ end (CYP71AZ1for 5′-ATGCAGATGGATGCAGTAGTTATCCTTCTTATTC-3′ and CYP71AZ1rev 5′-TTAGTGGTGATGGTGATGATGTGAAAACATATATG-3′). PCR conditions were as follows: 5 min at 98°C, 30 cycles (10 s at 98°C, 10 s at 55°C, and 120 s at 72°C), and a final 5 min extension step at 72°C. PCR products were purified, cloned using a pCR^®8/GW/TOPO^® TA Cloning^® Kit (Invitrogen^®), and sequenced. The resulting PCR product cloned in pCR^®8/GW/TOPO^® was sequenced and then subcloned in the yeast expression vector pYeDP60-GW (Dueholm et al., 2015 (link)) using LR clonase II (Invitrogen).

The overexpression pMDC32 binary vector49 was constructed as previously described [41 (link), 42 (link)]. The PaMYB10 coding sequence was PCR amplified from ‘JNL’ cDNA using the primers 5′-ATGGAGGGTTATTTCGGTGTGAG-3′ (forward) and 5′-TACGTAGGAGATGTTGACTAGATCATTGC-3′ (reverse) and cloned using the pCR™8/GW/TOPO TA Cloning Kit (Invitrogen). After sequencing confirmation, the CDS was recombined into the pMDC32 binary vector49 using the Gateway LR Clonase II Enzyme mix (Invitrogen) to produce overexpression vectors. The vectors were transformed into Agrobacterium tumefaciens GV3101 and then resuspended at a final O.D. of 0.8 in liquid MS medium. One milliliter of the suspension was evenly injected into the ‘LT’ fruit at commercial mature stage. Thirty fruits were injected as the repeat, and fruits were injected with Agrobacterium carrying an empty vector as a control treatment. At 7–12 days after injection, the fruit peel surrounding the injection sites was collected and immediately frozen in liquid nitrogen and stored at 80 °C for determining the anthocyanin according to the method of above mentioned.

Molecular biology experiments were performed according to standard procedures (44 ) and the supplier’s (Fermentas) recommendations. The NucleoBond Xtra Midi kit (Macherey-Nagel) and QIAamp DNA minikit (Qiagen) were used for plasmid preparations and H. pylori genomic DNA extractions, respectively. PCRs were performed with either Taq core DNA polymerase (MP Biomedicals) or with Phusion Hot Start DNA polymerase (Finnzymes) when the product required a high-fidelity polymerase. The PCR8/GW/TOPO TA cloning kit (Invitrogen) was used to construct in E. coli the suicide plasmid that served for complementation in H. pylori.

The coding sequences of SlGRXC9/Solyc08g036570, SlTGA9/Solyc06g074320 and SlTGA10/Solyc10g078670 were amplified by PCR and cloned into pCR8 using the pCR™8/GW/TOPO™ TA Cloning Kit (Invitrogen, now ThermoFisher Scientific). Then cDNAs were fused in frame at the N terminus with the N‐terminus half (nYFP) or C‐terminus half (cYFP) of the fluorescent YFP protein via Gateway. The vectors were transformed into Agrobacterium tumefaciens strain C58 and used to agroinfiltrate 4‐week‐old N. benthamiana leaves to a total OD₆₀₀ of 0.1. After 72 h of infiltration, protein–protein interactions were analyzed on leaf disks by confocal scanning microscopy (LSM 780; Zeiss). Intensity was adjusted using the negative control Del2GAI_cYFP (Gallego‐Bartolomé et al., 2012 (link)), which does not interact with SlTGA9, SlTGA10 or SlGRXC9.

Molecular biology experiments were performed according to standard procedures [71 ] and the supplier (Fermentas) recommendations. NucleoBond Xtra Midi Kit (Macherey-Nagel) and QIAamp DNA Mini Kit (Qiagen) were used for plasmid preparations and H. pylori genomic DNA extractions, respectively. PCR were performed either with Taq Core DNA polymerase (MP Biomedicals), or with Phusion Hot Start DNA polymerase (Finnzymes) when the product required high fidelity polymerase. The PCR8/GW/TOPO TA cloning kit (Invitrogen) was used to construct in E. coli, the H. pylori suicide plasmids that served for mutagenesis in H. pylori.